βig-h3 is an extracellular matrix protein whose expression is induced in various cell lines, including human melanoma cells, mammary ephithelial cells, keratinocytes, and lung fibroblasts, following signaling by active TGF-β. The βig-h3 gene was first isolated by differential hybridization screening of a cDNA library made from a human lung adenocarcima cell line that had been treated with TGF-β. βig-h3 gene encodes a 683-amino acid protein that is highly conserved between species. It contains an N-terminal secretory signal peptide and an Arg-Gly-Asp (RGD) motif at the C-terminus. The RGD motif, which modulates cell adhesion, is found in many extracellular matrix proteins and serves as a ligand recognition sequence for several integrins.
Because the expression of βig-h3 gene is increased by TGF-β in various cell lines and the gene is induced in various cell lines whose proliferation rate is controlled by TGF-β, βig-h3 is believed to be involved in mediating some of the signaling pathways of TGF-β. In contrast, βig-h3 expression is reported to be reduced in the fibroblasts cultured from the skin lesions afflicted with localized hyperostosis of melorheostosis, some tumor cells, and dexamethasone-treated stem cells. Accordingly, βig-h3 plays an important role in the morphogenesis and interactions with cells and other extracellular matrix proteins in various tissues.
Additionally, βig-h3 is known to mediate cell attachment and detachment, serving as a cell adhesion molecule. Purified βig-h3 protein is found to promote the attachment and spreading of skin fibroblasts while inhibiting the adhesion of A549, HeLa and Wi-38 cells in serum-free media. Particularly, βig-h3 is known to have inhibitory activity against tumor cell growth, and colony formation. In fact, it was reported that βig-h3 remarkably suppressed the growth of CHO (Chinese hamster ovary) cells in nude mice. Furthermore, a wound healing method was developed on the basis of the finding that application of a pharmaceutically effective amount of βig-h3 to wounds makes cells, especially fibroblasts, spread over and adhere to the wounded site. Consequently, βig-h3, a cell adhesion molecule induced by TGF-β in various cell lines, plays a very important roles in cell growth, cell differentiation, wound healing, morphogenesis and cell adhesion.
βig-h3 contains four 140 amino acid repeats with internal homology. The internally repeated domains have highly conserved sequences found in secretory proteins or membrane proteins of various species, including mammals, insects, sea urchin, plants, yeast and bacteria. Proteins such as periostin, fasciclin I, sea urchin HLC-2, algal-CAM and mycobacterium MPB70 are examples containing the conserved sequences. The homology domain conserved in these proteins (hereinafter referred to as “fas-1”) consists of about 110 to 140 amino acids with two highly conserved branches of H1 and H2 which have about 10 amino acids each. Four fas-1 domains are found in βig-h3, periostin, and fasciclin I, two fas-1 domains in HLC-2, and only one fas-1 domain in MPB70. Although the functions of the proteins are not elucidated clearly, some of them are known to act as cell adhesion molecules. For instance, βig-h3, periostin, and fasciclin 1 are reported to mediate the adhesion of fibroblasts, osteoblasts, and nerve cells, respectively. Also, it is disclosed that the algal-CAM is a cell adhesion molecule present in embryos of algae Volvox.
The cell attachment activity of βig-h3 was found first in human dermal fibroblasts and then in chondrocytes, peritoneal fibroblasts and human MRC5 fibroblast. At first, it was believed that the cell attachment activity of βig-h3 would be mediated by the C-terminal RGD motif. However, some research results revealed that the RGD motif is not necessary for promoting the spread of chondrocytes and that the mature soluble βig-h3 whose RGD motif is deleted by carboxyl-terminus processing is able to inhibit cell adhesion, leading to the conclusion that the RGD motif of βig-h3 is dispensable for mediating the cell attachment activity of βig-h3. In addition, it has been recently reported that βig-h3 promotes the spread of fibroblasts via integrin α1β1 whereas the RGD motif of βig-h3 is not necessary for βig-h3-mediated cell spreading. Further, the conserved peptides H1 and H2 of βig-h3 do not inhibit βig-h3-mediated cell adhesion, so that the conserved peptides are not effective for βig-h3-mediated cell attachment. These results, taken together, indicate that amino acids indispensable for the cell attachment activity of βig-h3 exist in regions other than H1 and H2. A computer analysis of the homology among fas-1 domains of other proteins as well as between repeated fas-1 domains of βig-h3 discloses the existence of several highly conserved amino acid sequences in addition to H1 and H2 peptides, suggesting the posibility of the involvement of the conserved amino acid sequences in the cell attachment activity.